In this research proposal, multinuclear (1H, 2H, 13C, 19F, and 31P) magnetic resonance spectroscopy is used as a means of studying (a) the nature of the conformation and bonding of selected folates and pyrimidine nucleotides at the active site of the enzyme thymidylate synthetase and (b) with a combination of creative biochemical investigations and multinuclear magnetic resonance studies we will also delineate the nature of the active site itself, i.e., the specific amino acid residues that are internally involved in the catalytic process occurring on the surface of thymidylate synthetase. Fourier transform assisted multinuclear magnetic resonance techniques are employed to obtain chemical shifts, relaxation times, and isotropic spin coupling constants which will be used to identify the loci (atoms or molecular groups) of the folates and pyrimidine nucleotides which are intimately involved in the binding of these compounds to thymidylate synthetase. To achieve this ambitious set of objectives we have developed a multinuclear 18 mm nmr probe which leads to an approximate increase in signal-to-noise ratio of 3.4 over conventional 12 nmr probes. To further aid these investigations we will employ isotopically enriched compounds already on hand from previous research projects. NMR procedures will also be used to characterize the behavior of thymidylate synthetase which has been chemically modified at specific sites with agents bearing an nmr sensitive nucleus or which has been obtained from bacteria cultured in the presence of selectively nmr-spin-labeled amino acids.